Epidemiological patterns and antimicrobial resistance of bacterial diarrhea among children in Nairobi City, Kenya

Aim: Determine the prevalence of enteric bacterial pathogens and their antimicrobial resistance among diarrheic children in Nairobi City, Kenya. Background: Regardless of enteric bacterial pathogens being a major cause of gastroenteritis in children, their occurrence and antimicrobial resistance patterns reveals regional spatial and temporal variation. Methods: In a cross-sectional study, a total of 374 children below five years presenting with diarrhea at Mbagathi County Hospital were recruited. Stool microbiology test was used to detect enteric bacterial infection. Antimicrobial resistance was determined using the disk diffusion method. Results: Diarrheagenic E. coli (36.4%) was the leading species followed by Shigella (3.2%), Salmonella (2.4%), Campylobacter (1.6%), Yersinia (1.3%) and Aeromonas (1.1%) species. Escherichia coli pathotyping revealed that 20.9%, 4.0%, 10.2% and 0.5% of the study participants were infected with enteroaggregative E. coli (EAEC), enteropathogenic E. coli (EPEC), enterotoxigenic E. coli (ETEC) and enteroinvasive E. coli (EIEC) pure isolates while the prevalence of mixed pathotype infections was 0.3% for EAEC/EPEC/ETEC and 0.5% for EAEC/ETEC. Shigella sero-grouping revealed that 0.5%, 0.3%, 1.9%, and 0.5% were infected with Shigella boydii, Shigella dysentriae, Shigella flexneri and Shigella sonnei pure isolates. Shigella species and E. coli co-infection was detected in 2.4% of the children, specifically, 1.1% for EAEC/Shigella boydii, 0.5% for EAEC/Shigella dysentriae and 0.3% in each case of EAEC/Shigella sonnei, EPEC/Shigella flexneri and ETEC/Shigella flexneri co-infections. Most of the isolates were resistant to commonly prescribed antibiotics. Conclusion: There was a high prevalence of enteric bacterial pathogens and co-infection alters epidemiological dynamics of bacterial diarrhea in children. Continuous antibiotic resistance surveillance is justified because the pathogens were highly resistant to commonly prescribed antimicrobials.


Introduction
In the year 2016, diarrhea accounted for more than 1.6 million deaths globally making it the fifth leading cause continents including Africa (1,2). In Sub-Saharan Africa, there were more than 1.2 billion cases of diarrhea, of which 371 million, leading to at least 0.2 million deaths, occurred in children younger than five years (1,2). Besides parasites and viruses, enteric bacteria continue to be a leading cause of gastroenteritis in children (1). Enteric bacterial pathogens accounts for more than 300 million episodes of diarrhea and one million deaths among children globally, the majority of which are caused by Escherichia coli and the remainder being Campylobacter, Shigella, Vibrio and Salmonella species (1). Previous studies across Kenya demonstrated that Shigella, E. coli, and Salmonella species were associated with childhood diarrhea (3), while others did not (4), suggesting geographic uniqueness of gut microbiota in immunity towards enteric infection patterns in Kenya (5)(6)(7)(8). Also, previous studies across Kenya found an association between Campylobacter and Shigella species with diarrhea among HIV infected children (9) while others did not (10), signifying that HIV alters the already geographic unique gut microbiota profile which is critical for host immunity to enteric bacterial infections (11,12). The risk factors of enteric bacterial pathogens, however, appear to be distributed differently (3,13,14) and as a result, the incidences of specific pathogens differ across Kenya (4,(14)(15)(16)(17)(18)(19)(20). A more recent study in Nairobi city identified Yersinia enterocolitica as an etiology of childhood diarrhea (3) while previous studies did not (13,17), indicating the emergence and spread of new pathogen strains driven by poor water, sanitation and hygiene (WASH) practices in the city (3) qualifying the need for constant epidemiological surveillance in Nairobi city, Kenya. However, there is paucity of information on the prevalence of enteric bacterial pathogens among diarrheic children in Nairobi city, Kenya. The innovation of antibiotics led to optimism that enteric bacterial infections could be controlled and prevented. However, enteric bacteria resistance to antibiotics is still the leading cause of death globally, including Kenya (21). Many studies have reported that enteropathogenic bacteria isolated from diarrheic children in Kenya including Nairobi city, can develop resistance (3,13,14,22,23). For instance, a previous study in Nairobi city reported the resistance of E. coli and Shigella isolates to ampicillin, trimethoprim/sulfamethoxazole, streptomycin, chloramphenicol and tetracycline (3) while a recent study showed changing patterns with increasing resistance (23). This increasing resistance is due to inappropriate antibiotic use increasing selection and transmission of antibiotic resistant strains in the city (3,13,22,24,25) in this era when there is a serious lack of new antibiotics under development to combat the growing antimicrobial resistance (26,27), justifying the need for continuous antimicrobial resistance surveillance. However, there is limited information on the prevalence of antimicrobial resistance of enteric bacterial pathogens among children in Nairobi city, Kenya. This study, therefore, aimed at determining the prevalence of enteric bacterial pathogens and their antimicrobial resistance among diarrheic children in Nairobi city, Kenya.

Study site, design and population
A detailed description of the study site, design and population is presented here (23). Briefly, this was a cross-sectional study targeting diarrheic children <5 years, seeking treatment at Mbagathi hospital, Nairobi city, Kenya. Diarrhea was defined according to World Health Organization (WHO) guidelines as the occurrence of three or more loose, liquid, or watery stools in a 24-hour period (28). Demographic and clinical information of the study participants were collected using a questionnaire. Stool specimens were collected and microbiology laboratory analysis performed within two hours of collection. Stool samples of children who had received antibiotics and who did not provide informed consent were excluded from the study.

Bacteriological procedures
Identification of bacteria species was performed according to WHO recommendations (29). All fecal specimens were cultured in alkaline peptone water, selenite broth and blood agar enrichment media, followed by sub-culture on selective and differential media as described elsewhere (18). Isolates were then subjected to Gram staining and biochemical tests using oxidase test, lysine decarboxylase test, urease test, citrate test, hydrogen sulfide gas production, fermentation test, and motility test to identify the significant characteristic of bacteria according to the standard methods (29). E. coli pathotyping and Shigella sero-grouping was performed as previously described (23).

Antimicrobial Resistance
Antibiotic susceptibility was performed using Kirby-Bauer disk diffusion method on Mueller Hinton agar by incubating at 37°C for 18 hours (30). Broth turbidity was made to match with 0.5 McFarland standards. Antibiotics discs of ampicillin, trimethoprim/sulfamethoxazole, ceftriaxone, streptomycin, amoxicillin/clavulanic acid, gentamycin, kanamycin, ciprofloxacin, chloramphenicol, erythromycin, nalidixic acid and tetracycline were tested.

Data analysis
Statistical analyses were performed using SPSS version 19.0 for Windows (IBM SPSS Statistics for Windows, Version 19.0. Armonk, NY: IBM Corp.). Descriptive statistics, namely frequencies and percentages, were used to present demographic and clinical data, frequency of enteric bacteria pathogens and their antimicrobial susceptibility pattern.

Ethical considerations
This study was ethically approved by Kenyatta National Hospital/University of Nairobi (KNH-UoN) Ethics and Research Committee and was conducted according to the Declaration of Helsinki (31). A consent form was read and signed by either parent or guardian of each child. Diarrheic children were treated by clinicians according to World Health Organization (WHO) guidelines for treatment of diarrhea in children (28). All study participants' information and test results were kept confidential. The results of bacterial cultures were used in clinical management of study participants.

Discussion
Globally, enteric bacteria associated diarrhea continues to be a major cause of morbidity and mortality among children under 5 years (1). Enteric bacterial pathogens epidemiology shows variations between countries, and even between geographical regions within the same country (32). There has been a dramatic increase in the emergence of antimicrobial resistant enteric bacterial strains, which has made antibiotic choices for enteric infection treatment increasingly limited and more expensive (21). Thus, continuous epidemiological and antimicrobial resistance surveillances are fundamental in planning treatment. Diarrheagenic E. coli predominates in this study followed by the rest namely Shigella, Salmonella, Campylobacter, Yersinia and Aeromonus species, in that order, highlighting the prominent role of diarrhegenic E. coli in enterobacteria associated diarrhea among children in Nairobi city, Kenya. These findings are consistent with previous studies involving diarrheic children in Kiambu (18) and Homa Bay counties, Kenya (19). As reviewed by (33), diarrhegenic E. coli persists longer in environmental reservoirs supporting the hypothesis of increased transmission of E. coli to human population (34), reinforcing the observation of this study. However, the findings of the present study are inconsistent with previous studies in the literature that demonstrated the dominancy of Shigella species, followed by diarrhegenic E. coli, Salmonella and Yersinia species among diarrheic children in Nairobi city, Kenya (3). This may be attributed to the fact that  proved diarrhegenic E. coli, particularly EAEC, genome heterogeneity (35), using two virulence genes for EAEC detection decreased the rate of isolation in the previous study (3). Above all, this study detected higher prevalence of Shigella mono-infection (3.2%) than Shigella/E. coli co-infection (2.4%) suggesting that coinfections alter epidemiological dynamics of infectious disease due to pathogen synergism worsening disease severity and consequent mortality (36), and this observation is consistent with previous studies among diarrheic children in Kiambu county, Kenya (19), China (37), Zanzibar and Rwanda (38). Therefore, diarrhegenic E. coli, Shigella, Salmonella, Campylobacter, Yersinia and Aeromonas species are principal causes of childhood bacteria associated diarrhea justifying access to safe water, sanitation and hygiene among children in Nairobi city, Kenya. The current study observed high antimicrobial resistance rates indicating rapid and ongoing spread of antimicrobial-resistant organisms. Specifically, more than half of diarrheagenic E. coli isolates were resistant to ampicillin, trimethoprim/ sulfamethoxazole, streptomycin, kanamycin, chloramphenicol and tetracycline which is partly in agreement with studies in Nairobi city (17) and Meru county (14) but inconsistent with a previous study among the Maasai community of Narok and Kajiado counties that reported increased susceptibility to these antibiotics (39), possibly due to reduced antimicrobial use among the Maasai community who mostly practice traditional medicine (40). Over half of the Shigella isolates were resistant to streptomycin, chloramphenicol and tetracycline while resistance to ampicillin, ceftriaxone, amoxicillin-clavulanic acid, kanamycin, ciprofloxacin, and erythromycin occurred in more than half of Salmonella isolates, which partly agrees with a multisite study conducted in Kisii, Homa Bay and Migori counties, Kenya (22) but conflicts with a study in Zambia (41). Over half of Campylobacter isolates were resistant to ampicillin, chloramphenicol, and erythromycin which is consistent with the findings in Ethiopia (42) and disagrees with a study in China (43). At least half of Yersinia isolates were resistant to ampicillin, streptomycin, gentamycin and tetracycline which mirrors a study in Ethiopia (44) but disagrees with a study in Mexico (45). At least half of Aeromonas isolates were resistant to tetracycline and chloramphenicol which is in agreement with a study in Brazil (46) and disagrees with a study in China (43). These variations in resistance profiles between countries, within regions suggest significant differences in antibiotic use (47). Therefore, antimicrobial stewardship programs have to be developed to influence antibiotic use and prescribing behavior to ensure longterm availability of effective treatment for bacterial infections in Kenya. This study had limitations. Other studies have detected viral, bacterial and parasitic gastroenteritis among Kenyan children (3,4,19) thus it is possible that the bacterial pathogens found may not be the sole cause of the diarrhea. Study participants were recruited within the hospital hence the prevalence of enteric bacteria and antimicrobial resistance does not represent community prevalence. We did not assay antimicrobial resistance genes. The presence of DAEC and AIEC pathotypes were not investigated in this study. We acknowledge the small sample size of Aeromonas, Yersinia and Campylobacter isolates assayed for antimicrobial resistance. We conclude that diarrhegenic E. coli, Shigella, Salmonella, Campylobacter, Yersinia and Aeromonas species are important etiologies of diarrhea in children under five years of age, in Kenya. These pathogens are of public health importance since they are highly resistant to commonly prescribed antimicrobials. It is hoped the results of the present study will guide treatment of bacterial diarrheal diseases among children in the country.